1. Field of the Invention
This invention relates generally to the field of nucleic acid chemistry. More specifically, it relates to methods of decreasing the fluorescence of fluorescently labeled oligonucleotides in solution using a DNA binding compound. Additionally, it relates to methods for detecting degradation of fluorescently labeled single-stranded oligonucleotides in solution. Additionally, the invention relates to methods for detecting nucleic acid sequences by hybridization with a complementary oligonucleotide probe.
2. Description of the Related Art
Nucleic acid detection using oligonucleotide probes has become a standard method for specific target detection. Numerous assay formats have been described. Generally, a nucleic acid sample is hybridized to a labeled target-specific probe, unbound probe is separated from the hybridization duplexes, and the presence of hybridization duplexes are detected using the label. Separation of the hybridized and unhybridized probes can be achieved by a number of means. For example, either the sample nucleic acid may be immobilized on a solid support and the unhybridized probe removed by washing, or the hybridization duplexes and unbound probe may be separated by gel electrophoresis. In general, the methods require a separation step in order that the signal generated following hybridization can be distinguished from the background signal generated by the unbound labeled probe.
Several nucleic acid detection methods have been described which involve selective cleavage of oligonucleotide probes following formation of probe-target hybridization duplexes. Detection of cleaved probes indicates the occurrence of hybridization and, hence, the pence of target sequences. For example, Saiki et al., 1985, Biotechnology 3:1008-1012, incorporated herein by reference, describe "oligomer restriction" detection methods, in which hybridization of the target-specific probe generates a restriction site which is then cleaved by the corresponding restriction enzyme. PCT Patent Publication No. WO 89109284, incorporated herein by reference, describes methods in which RNA probes are used to detect DNA target sequences. RNA probes hybridized to DNA target are cleaved using RNaseH, which selectively cleaves RNA in RNA-DNA hybrid duplexes. U.S. Pat. No. 5,210,015, incorporated herein by reference, describes methods which use the 5' to 3' exonuclease activity of a nucleic acid polymerase to cleave probes hybridized to target sequences and thereby release labeled oligonucleotide fragments for detection.
The invention of the polymerase chain reaction (PCR), a process for amplifying nucleic acids, enabled the detection of nucleic acids with greatly increased sensitivity and specificity. Using PCR, segments of single copy genomic DNA can be selectively amplified to an easily detectable level prior to detection. PCR methods are disclosed in U.S. Pat. No. 4,683,202, incorporated herein by reference. PCR and methods for detecting PCR products using an oligonucleotide probe capable of hybridizing with the amplified target nucleic acid are described in U.S. Pat. No. 4,683,195, and European Patent Publication No. 237,362, both incorporated herein by reference.
The methods for detecting nucleic acid described above which involve selective cleavage of hybridization probes following formation of probe-target hybridization duplexes have been applied to the detection of amplified nucleic acid. Saiki et al., 1985, Science 230:1350-1353, incorporated herein by reference, describe the application of "oligomer restriction" to the detection of PCR amplified nucleic acid. U.S. Pat. No. 5,210,015, supra, describes the analysis of PCR amplification products using the 5' to 3' exonuclease activity of a nucleic acid polymerase to cleave labeled probes hybridized to target sequences (see also Holland et al, 1991, Proc. Natl. Acad. Sci. USA 88:7276-7280, incorporated herein by reference). In the methods of the '015 patent, probes that hybridize to a region of the target nucleic acid bounded by the amplification primers are incorporated into the amplification reaction mixture. Hybridized probes are cleaved by the 5' to 3' nuclease activity of the polymerase during primer extension. Detection of labeled fragments indicates the occurrence of both primer extension and probe hybridization, and, therefore, amplification of the specific target sequence.
A number of agents have been described for labeling nucleic acids, whether probe or target, for facilitating detection of target nucleic acid. Labels have been described that provide signals detectable by fluorescence, radioactivity, colorimetry, X-ray diffraction or absorption, magnetism, and enzymatic activity and include, for example, fluorophores, chromophores, radioactive isotopes (particularly .sup.32 P and .sup.125 I), electron-dense reagents, enzymes, and ligands having specific binding partners. Labeling can be achieved by a number of means, such as chemical modification of a primer or probe to incorporate a label or the use of polymerizing agents to incorporate a modified nucleoside triphosphate into an extension product.
The use of oligonucleotide probes labeled with interacting fluorescent labels in nucleic acid hybridization assays is described in Morrison, 1992, in Nonisotopic DNA Probe Techniques, Kricka, ed., Academic Press, Inc., San Diego, Calif., chapter 13; and Heller and Morrison, 1985, in Rapid Detection and Identification of Infectious Agents, Academic Press, Inc., San Diego, Calif., pages 245-256; both incorporated herein by reference. The methods rely on the change in fluorescence that occurs when suitable fluorescent labels are brought into close proximity, described in the literature as fluorescence energy transfer (FET), fluorescence resonance energy transfer, nonradiative energy transfer, long-range energy transfer, dipole-coupled energy transfer, or F orster energy transfer.
Morrison, 1992, supra, describes three assay formats. In two of the assay formats, interacting fluorescent labels are bound to separate oligonucleotides that are either brought together or separated by probe hybridization. These assay formats are described as either non-competitive or competitive, depending on whether probe-probe hybridization competes with probe-target hybridization. Both assays formats require the synthesis of two sequence-specific labeled probes. In the third assay format, one fluorescent label is bound to the hybridization probe, and the second fluorescent label is brought into close proximity by intercalating into the double-stranded hybridization duplex. No significant interaction occurs between the intercalating label and the unhybridized probe in solution. Because the intercalating label can intercalate into any double-stranded nucleic acid, this format is practical only for the detection of single stranded target nucleic acid.
U.S. Pat. No. 5,210,015, supra, describes the use of a hybridization probe that is labeled with interacting fluorescent labels in close proximity. The labels are attached such that probe degradation during amplification separates the labels, thereby producing a detectable change in fluorescence. Such multiply-labeled probes are difficult and costly to synthesize.
Conventional techniques of molecular biology and nucleic acid chemistry, which are within the skill of the art, are fully explained fully in the literature. See, for example, Sambrook et al., 1985, Molecular Cloning--A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.; Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Nucleic Aria Hybridization (B. D. Hames and S. J. Higgins. eds., 1984); and a series, Methods in Enzymology (Academic Press, Inc.), all of which are incorporated herein by reference. All patents, patent applications, and publications mentioned herein, both supra and infra, are incorporated herein by reference.